Recombinant proteins are produced in living cells and are a major class of biologic drugs used to treat a wide range of diseases. Production of recombinant proteins in cell culture is complicated by the fact that a cell's host proteins can modify recombinant proteins by adding a variety of PTMs, such as glycosylation, carboxylation, hydroxylation, sulfation and amidation, deamidation, among others. Some examples of inhibitors are shown in FIG. 1. Some host proteins, including but not limited to Carboxypeptidase B, can even modify recombinant proteins by removing amino acids from the sequence, such as C-terminal lysines. PTMs are very difficult to control during production of recombinant proteins, especially if a specific PTM is required; such as during biosimilar production or when making manufacturing process changes.
The goal of biosimilar development is to produce a biosimilar product with a similar and ideally identical PTM profile to a reference product or protein of interest. The capability of producing a recombinant protein copy that displays minimal or no differences from a marketed reference product can simplify the clinical testing required for drug approval and should reduce the cost of biosimilar drug development. One major reason for the more limited requirement for clinical studies with biosimilars is that the safety risks are known when product attributes are the same between a biosimilar and its reference product. Large hurdles in biosimilar development are related to the fact the production process for many commercial biologic drugs is either not known, may be protected by patents, or is outdated (i.e. use of animal derived products).
Thus, it is necessary to develop a production process that may be different from the original process but that still yields a copy of the reference product (biosimilar) with the same, or similar PTMs as the reference product.
Similarly, during a manufacturing process change, the ability to match PTMs on a recombinant protein before and after process changes is of uttermost importance. The key reason for a process change for a recombinant protein is to improve the cell line performance, to increase productivity, and to increase cell stability without changing PTMs of said recombinant protein. A method is needed that can decouple the optimization of productivity of the cell line from optimization of PTMs.
In order to develop such a method the host proteins that need to be modulated must be identified and modulators necessary to modulate those host proteins are then selected. Host proteins include enzymes involved in glycosylation, carboxylation, hydroxylation, deamidation, oxidation, C-terminal sulfation, C-terminal carboxylase and amidation or any other posttranslational modification. Modifying the activity of these enzymes using small molecules, natural products, biologics, RNAi, RNA, or DNA can be used for production of a recombinant protein with target PTMs. A method that is capable of modifying PTMs on biologic molecules would be far superior for use in the production of biosimilar and biobetter biologics than known systems that knock-out PTMs altogether. Such systems could be used to produce biologic molecules having PTMs within target ranges as opposed to knock out technologies which have no possibility of targeting a desired PTM range.